Current antipsychotic therapies can address the positive symptoms, but the negative and cognitive symptoms remain poorly managed, if at all, and are key predictors of functional disability. A large number of anatomical, molecular, genetic, preclinical behavioral and human clinical studies have provided strong evidence that agents able to enhance cholinergic transmission or activate muscarinic acetylcholine receptors (mAChRs, M1- M5), notably M1, have exciting therapeutic potential for the treatment of the positive, negative and cognitive symptoms of schizophrenia as well as cognitive dysfunction in other CNS disorders. However, previous compounds developed to selectively activate M1 receptors have failed in clinical development due to a lack of true specificity for M1 and adverse effects associated with activation of other mAChR subtypes (M2-M5). Furthermore, the lack of highly selective compounds has made it impossible to definitively determine whether the behavioral and clinical effects of these compounds are mediated by M1. Mechanistically, it is intriguing that selective M1 activation fits well with the glutamate hypothesis, or NMDA (N-methyl-D-aspartate) hypofunction hypothesis of schizophrenia, as both M1 and the NR1a NMDA subunit are co-localized at specific postsynaptic sites, and activation of M1 by orthosteric mAChR agonists, and more recently, highly selective M1 allosteric agonists and M1 positive allosteric modulators (PAMs), developed during the previous funding period, potentiate NMDA currents in hippocampal slices, display robust efficacy in preclinical antipsychotic rodent models and improve cognitive performance in multiple hippocampal-driven models. When this grant was initially funded four years ago, no truly selective M1 activators existed with the requisite mAChR selectivity, ancillary pharmacology or DMPK properties to study the role of selective M1 activation in vivo. Thus, during the previous funding period, we were highly successful in developing a vast array of highly selective M1 activators (both functionally M1 selective agonists and M1 PAMs representing multiple chemotypes) with unprecedented, clean ancillary pharmacology and DMPK profiles enabling in vivo studies to be performed via the chemical optimization of hits from a functional M1 high-throughput screen. Based on ligand-biased signaling phenomena, in order to definitively evaluate selective M1 activation in the context of NMDA hypofunction models of schizophrenia, we must develop a suite of M1 ligands with comparable DMPK properties that activate all M1-mediated pathways, as well as select, specific pathways in vivo. This research has direct relevance to the mission of NIMH and has the potential to impact human health directly. Our goal for this project is to develop, in parallel, selective M1 allosteric agonists and M1 positive allosteric modulators with acceptable profiles for preclinical and ultimately clinical development that may lead to a new drug for the treatment of the positive, negative and cognitive symptoms of schizophrenia.